There exists a conspicuous disparity in the lateral (i.e., x, y) and axial (i.e., z) imaging speed in conventional optical microscopy. While an image at the x-y plane can be routinely acquired in parallel or at high speed, slow mechanical scanning of the objective lens or the specimen itself is typically required in the z direction for axial imaging. This hinders the ability to image fast processes in the axial direction.
Laser scanning optical microscopy, such as multi-photon and confocal microscopy, has become a widely used imaging modality due to its unique optical sectioning capability. However, slow scanning, particularly in the axial (or z) direction remains a hindrance. This typically requires mechanical axial scanning of the specimen or the excitation objective lens, limiting the z-imaging speed and hence the ability to visualize fast dynamic processes in the axial slice plane and overall in the three dimensions.
In an effort to address this challenge, multi-focal imaging has been investigated in wide-field microscopy by using multiple cameras with each placed at a different distance to image a different conjugate plane. Holography has also been applied to fluorescence microscopy to achieve three-dimensional imaging. However, these techniques lack optical sectioning capability.
Methods to achieve multi-focal imaging in scanning microscopy include time and wavelength division multiplexing. In a one-time division multiplexing technique, two sequences of excitation pulses with different wave-front divergence (thereby to focus on two different depth positions) are interleaved in time. The fluorescence signal generated at the two depth positions can then be de-multiplexed in time to realize bi-focal imaging. In epi-reflection chromatic confocal microscopy different wavelengths of a broadband source are focused onto different axial positions inside a specimen through purposely-introduced chromatic aberration. Light reflected from different axial positions has different wavelengths and can therefore be detected in parallel by using a spectrometer.
This method has also been extended to second harmonic microscopy, in which different fundamental wavelengths of a pump pulse are focused to different axial positions to produce second harmonic signals of different center wavelengths that can be simultaneously detected. However, parallel z-imaging has yet to be realized using wavelength division multiplexing in scanning fluorescence microscopy, one of the most widely used imaging modalities. Although chromatic scanning can be utilized to accomplish effective axial scanning, the fluorescence signals excited at different axial positions exhibit similar emission spectra, preventing the use of parallel spectroscopic detection.